Combination of immune effector cells specific for a target antigen and hematopoietic cells that express the target antigen in an altered form

ABSTRACT

The invention provides a system that comprises pharmaceutical agents for use in immunotherapy for reducing the side-effects of an antigen-recognizing receptor against antigen-expressing non-target cells in an individual. The system includes an antigen-recognizing receptor that specifically recognizes an antigen on target cells and at least on one hematopoietic cell type in the individual. The antigen-recognizing receptor is exemplified by chimeric antigen receptors (CAR) be expressed on the surface of an immune effector cells. The system also includes hematopoietic cells resistant to recognition of the same antigen by the antigen-recognizing receptor.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/952,448, filed Nov. 25, 2015 (pending) which claims the prioritybenefit of DE 102014224071.9, filed Nov. 26, 2014. The aforelistedpriority applications are hereby incorporated herein by reference intheir entirety for all purposes.

FIELD OF INVENTION

This present invention relates to the treatment of diseases such ascancer using antigen-recognizing receptors, e.g. chimeric antigenreceptors (CARs) on immune effector cells in combination with thetransfer of polymorphic or genetically modified hematopoietic cells tocircumvent or reduce side-effects of said antigen-recognizing receptor.

BACKGROUND

Targeted immunotherapies are based on the recognition of antigens,defined structures on diseased cells or pathogens, by immune receptorsthat are either soluble or present on the surface of immune cells.Recognition and binding of the antigen by the immune receptor usuallytriggers effector functions that eventually lead to the destruction ofthe respective pathogen or cell. Soluble immune receptors includenatural or synthetic antibodies, antibody derived molecules and otherstructures, which upon binding to an antigen trigger the complementsystem or recruit and in most cases activate effector cells. Directtriggering of cell effector function can be induced upon antigenrecognition by cell membrane bound immune receptors such as T cellreceptors (TCR) present on T lymphocytes. TCRs specifically recognizeantigenic peptides that are presented on human leukocyte antigen (HLA)molecules on virus infected or malignant cells, which leads toactivation and T cell mediated killing of target cells. Antigen-specificT cells of the in vivo occurring natural repertoire can be applied fortherapeutic purposes using various methods. Alternativelyantigen-targeting cells can be generated through the genetic insertionof engineered immune receptors, such as transgenic TCRs or CARs into Tcells or other immune effector cells including natural killer (NK)cells. Commonly, CARs comprise a single chain fragment variable (scFv)of an antibody specific for a certain target antigen coupled via hingeand transmembrane regions to cytoplasmic domains of T-cell signalingmolecules. The most common lymphocyte activation moieties include a Tcell co-stimulatory (e.g. CD28, CD137, OX40, ICOS, and CD27) domain intandem with a T cell triggering (e.g. CD3ζ) moiety. The CAR-mediatedadoptive immunotherapy allows CAR-grafted cells to directly recognizethe desired antigen on target cells in a non-HLA-restricted manner.

Cancer is a broad group of diseases involving deregulated cell growth.In cancer, cells divide and grow uncontrollably, forming malignanttumors, and invading nearby parts of the body. The cancer may alsospread to more distant parts of the body through the lymphatic system orbloodstream. There are over 200 different known cancers that affecthumans. Whereas good treatment options are available for many cancertypes, others still represent unmet medical needs. Cancers of thehematopoietic system can be roughly divided into different subtypes.Leukemias generally affect the primary lymphatic organs, which are thebone marrow as well as the thymus, and arise from hematopoieticprogenitor populations. Lymphomas on the other hand are usually derivedfrom mature lymphocytes and originate from secondary lymphatic organs.The current first line treatment for most hematopoietic cancers involvesthe administration of chemotherapeutic agents, radiation therapy or acombination of both. In many cases such therapies are combined with orfollowed by hematopoietic stem cell transfer (HSCT), where the graftversus leukemia (GVL) effect mediated by donor-derived lymphocytes,especially T cells, can lead to the eradication of cancer cells thatsurvived pre-conditioning chemo- or radiotherapies and result incomplete remission. Depending on the type of hematological malignancy,the patients' condition and the availability of hematopoietic stem cellgrafts various versions of HSCT are regularly performed in the clinics.The desired GVL effect is only achieved in allogeneic HSCT, which at thesame time is often accompanied by the occurrence of graft versus hostdisease (GvHD), a serious and sometimes fatal complication. Moreover, inall cases, persisting cancer stem cells often lead to disease relapse.

The CAR provides a promising approach for adoptive cell immunotherapyfor cancer. CAR T cell therapy has been tested for the treatment ofvarious cancers, hematopoietic cancers but also tumors derived fromother tissues, in clinical or pre-clinical studies and CARS for multipledifferent target antigens have been evaluated (Anurathapan, U., Leen, A.M., Brenner, M. K., and Vera, J. F. (2014). Engineered T cells forcancer treatment. Cytotherapy 16, 713-733.). Amongst hematologicalcancers, CAR modified autologous T cells present a promising tool toimprove the GVL effect without the complication of GvHD. As such CARshave been applied most successfully for the treatment of B-cell derivedcancers such as ALL and CLL using CD19 as target antigen (Grupp, S. A.,Kalos, M., Barrett, D., Aplenc, R., Porter, D. L., Rheingold, S. R.,Teachey, D. T., Chew, A., Hauck, B., Wright, J. F., et al. (2013).Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.The New England journal of medicine 368, 1509-1518. Porter, D. L.,Levine, B. L., Kalos, M., Bagg, A., and June, C. H. (2011). Chimericantigen receptor-modified T cells in chronic lymphoid leukemia. The NewEngland journal of medicine 365, 725-733.). However, for various othercancers and target antigens so called on-target off-tumour side effectshave been observed (Morgan, R. A., Yang, J. C., Kitano, M., Dudley, M.E., Laurencot, C. M., and Rosenberg, S. A. (2010). Case report of aserious adverse event following the administration of T cells transducedwith a chimeric antigen receptor recognizing ERBB2. Molecular therapy18, 843-851.). In these cases the CAR T cells specifically targeted thedesired antigen but the target antigen expression was not restricted tothe cancer. Consequently healthy tissues were damaged, which in somecases led to serious side effects or even death. Therefore theapplication of CAR T cell therapy to a wider range of cancers has beenhampered by the lack of suitable target antigens.

Antigen specific T cells can be used in combination with HSCT. Inmultiple clinical trials patients with lymphoid leukemias were treatedwith CAR T cells specific for CD19 in order to achieve a temporaryremission and bridge the time until the identification of a suitabledonor for HSCT (Brentjens, R. J., Davila, M. L., Riviere, I., Park, J.,Wang, X., Cowell, L. G., Bartido, S., Stefanski, J., Taylor, C.,Olszewska, M., et al. (2013). CD19-targeted T cells rapidly inducemolecular remissions in adults with chemotherapy-refractory acutelymphoblastic leukemia. Science translational medicine 5, 177ra138.). Asimilar approach has been proposed for a CAR directed to CD123, anantigen that is expressed by myeloid leukemias, but also present on thehealthy myeloid cell compartment (Gill, S., Tasian, S. K., Ruella, M.,Shestova, O, Li, Y., Porter, D. L., Carroll, M., Danet-Desnoyers, G.,Scholler, J., Grupp, S. A., et al. (2014). Preclinical targeting ofhuman acute myeloid leukemia and myeloablation using chimeric antigenreceptor-modified T cells. Blood 123, 2343-2354.). The myeloid depletionefficiency has been shown for a CD123 CAR in a pre-clinical modelsuggesting that CD123 CAR T cells could be used as part of apre-conditioning regiment before HSCT. However, in order to avoidon-target off-tumour toxicity CAR T cells have to be completely absentat the time of HSCT as well as thereafter. For the CD19 CAR approach theapplication of CAR T cells and the HSCT might be month apart and someCD19 CAR T cells have been shown to have limited life spans (Brentjens,R. J., Davila, M. L., Riviere, I., Park, J., Wang, X., Cowell, L. G.,Bartido, S., Stefanski, J., Taylor, C., Olszewska, M., et al. (2013).CD19-targeted T cells rapidly induce molecular remissions in adults withchemotherapy-refractory acute lymphoblastic leukemia. Sciencetranslational medicine 5, 177ra138.). For the CD123 CAR, however, CAR Tcell treatment has to be followed by HSCT within days to avoid potentialmyelocytopenia and rapid CAR T cell depletion presents a significantchallenge.

For engineered T cells expressing a CAR or a transgenic TCR on-targetoff-tumor side effects can also include the so called T cell fratricide,if the target antigen is expressed by the T cells themselves. For a CD38CAR T cell fratricide observed during in vitro culture could beprevented using an anti-CD38 antibody that blocked the CAR-targetinteraction. Such approach, however, has to date not been tested invivo. Antibody mediated blocking of fratricide in vivo has only beenshown for NK cells in a murine model using a monoclonal antibody againstCD244 (Taniguchi, R. T., Guzior, D., and Kumar, V. (2007). 2B4 inhibitsNK-cell fratricide. Blood 110, 2020-2023.).

In a clinical safety study using a CAIX CAR for the treatment of renalcell carcinoma on-target off-tumor toxicity was reported due to lowtarget antigen expression in the liver (Lamers, C. H., Sleijfer, S., vanSteenbergen, S., van Elzakker, P., van Krimpen, B., Groot, C., Vulto,A., den Bakker, M., Oosterwijk, E., Debets, R., et al. (2013). Treatmentof metastatic renal cell carcinoma with CAIX CAR-engineered T cells:clinical evaluation and management of on-target toxicity. Moleculartherapy 21, 904-912.). On-target off-tumor toxicity could be preventedby treatment with a CAIX monoclonal antibody, which blocked theCAR-target interaction. However, with antibody treatment the anti-tumorresponse was equally undetectable.

For T cells expressing a transgenic TCR, fratricide can potentially becircumvented, if an allogeneic T cell donor negative for the targetedHLA-type is used (Leisegang, M., Wilde, S., Spranger, S., Milosevic, S.,Frankenberger, B., Uckert, W., and Schendel, D. J. (2010).MHC-restricted fratricide of human lymphocytes expressingsurvivin-specific transgenic T cell receptors. The Journal of clinicalinvestigation 120, 3869-3877. Schendel, D. J., and Frankenberger, B.(2013). Limitations for TCR gene therapy by MHC-restricted fratricideand TCR-mediated hematopoietic stem cell toxicity. Oncoimmunology 2,e22410).

Genetic modification of HLA molecules in order to create cells that areno longer the target of particular transgenic or naturally occurringTCRs is disclosed in WO2012012667A2. Designer nucleases such as ZFNs orTALENs are applied for the deletion of one or more HLA molecules.

Designer nuclease mediated modification of hematopoietic cells includingT cells and hematopoietic stem cells (HSC) has been described for thegeneration of HIV resistant T cells (Holt, N., Wang, J., Kim, K.,Friedman, G., Wang, X., Taupin, V., Crooks, G. M., Kohn, D. B., Gregory,P. D., Holmes, M. C., et al. (2010). Human hematopoietic stem/progenitorcells modified by zinc-finger nucleases targeted to CCR5 control HIV-1in vivo. Nature biotechnology 28, 839-847.) (US20120309091A1). CCR5, aco-receptor required for HIV entry into T cells, is deleted using a ZFN.Generated CCR5 deleted T cells have been shown to be insensitive to HIVinfection.

The development of designer nucleases (ZFN, TALEN and CRISPR/Cas) aswell as transcriptional repressors (zinc finger, TALE- orCRISPR/Cas-based fusion proteins) for the deletion or transcriptionalrepression of the hematopoietic surface proteins CTLA-4 and PD-1,optionally in combination with CARs or transgenic TCRs, has beendisclosed in WO2014059173A2.

Taken together, in recent years there has been strong progress in thedevelopment of targeted immunotherapies for multiple diseases includingsome types of cancer. However, the lack of suitable target molecules fortherapies with antigen-recognizing receptors and in particular CAR Tcells has been a major obstacle. Therefore, there is a need for thedevelopment of novel therapies for the treatment of diseases, such ascancer, that enable the utilization of alternative target molecules, andreduce or avoid the side-effects often associated with current targetedimmunotherapies in general, and CAR T cell therapies in particular.

SUMMARY OF THE INVENTION

Surprisingly, it was found that the side-effects of anantigen-recognizing receptor, which recognizes an antigen present ontarget cells, but also on at least one hematopoietic cell type, can bereduced by application of hematopoietic cells resistant to recognitionby said antigen recognizing receptor in a combination immunotherapy.Therefore this invention relates to a combination immunotherapy fordisease in an individual, including but not restricted to cancer,comprising i) an antigen-recognizing receptor, which recognizes anantigen present on target cells, but also on at least one hematopoieticcell type of said individual, and ii) hematopoietic cells resistant torecognition of said antigen by said antigen-recognizing receptor.

As an effect of the present invention a reduction of side-effects ofantigen-recognizing receptors by application of hematopoietic cellsresistant to recognition by said antigen-recognizing receptor in acombination therapy can be observed. Furthermore the invention relatesto a method or system that allows the utilization of a group of antigensas potential targets for immunotherapy, that are present on at least onehematopoietic cell type, and therefore are not suitable targets forimmunotherapies currently known in the art. In one embodiment saidhematopoietic cells resistant to recognition of said antigen-recognizingreceptor express a naturally occurring version of said antigen. Inanother embodiment said hematopoietic cells resistant to recognition ofsaid antigen-recognizing receptor are genetically modified to express analtered version of said antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of a preferred embodiment of theinvention, detailing the application of immune effector cells expressingan antigen-recognizing receptor and hematopoietic cells resistant torecognition of said antigen by said antigen recognizing receptor to anindividual.

FIG. 2: Expression of human CD20 WT (SEQ ID NO:3) and CD20 mutantA170S/P172S. HEK 293T cells were either mock transfected or transfectedusing the pMACS LNGFR-IRES plasmid (Miltenyi Biotec GmbH) encodingeither wild type CD20 (annotated CD20 WT) or a mutated CD20 (CD20A170S/P172S). Two days after transfection, the HEK 2913T cells wereanalysed by Flow cytometry for detection of the CD20 antigen usingdifferent anti-CD20 antibodies (staining of CD20 extracellular epitopeswith clone LT20 and clone 2H7 and intracellular staining of CD20C-terminal epitope with clone 1412).

FIG. 3: Cytokine expression of human T cells modified to express anantigen-recognizing receptor directed against the CD20 antigen(annotated T-CAR) or human T cells not engineered to express anantigen-recognizing receptor (annotated T-MOCK) co-cultured with HEK 293T cells encoding either wild type CD20 (annotated 293T-CD20) or amutated CD20 A170S/P172S (annotated 293T-CD20Mut) or no antigen(annotated 293T-Mock). Results for the expression of either GM-CSF (leftgraphs) or IL-2 (right graphs) for T cells derived from 2 blood donorsare represented. Cytokines were measured from the supernatant after 24hour co-culture using the MACSPlex Cytokine kit of Miltenyi Biotec.X-axis represents the different conditions. A and B indicateexperimental duplicates.

FIG. 4: Gene editing of the CD20 antigen using designer nucleases inorder to abrogate recognition of the antigen by the antigen recognizinganti-CD20 antibody. The B cell lymphoma cell line Raji, naturallyexpressing CD20, was transfected by co-electroporation with 3 plasmids;one encoding the green fluorescent protein (as transfection control),one encoding the guide RNA sequences (gRNA 1-3) (see SEQ ID NO:4 and SEQID NO:5, SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,respectively) and one encoding the nuclease Cas9. 5 days aftergene-editing, the CD20 expression of the GFP positive Raji cells wasanalyzed by flow cytometry using the anti-CD20 antibody used to generatethe CAR . The 2 dot plots on the right represent controls as indicated.

FIG. 5: Table from the manuscript of Zebedee, S. L.; Barritt, D. S.; andRaschke, W. C. (1991. Comparison of Mouse Ly5a and Ly5b Leucocyte CommonAntigen Alleles. Developmental Immunology, 1, 243-254) showing thecomparison of sequence change between Ly5a and Ly5b which is mouseCD45.1 and CD45.2 isoforms. Thanks to the existence of 2 separateantibodies (anti-CD45.2 and anti-CD45.1), both isoforms can be detectedand discriminated.

FIG. 6: Flow cytometry analysis of HEK 293 T cells transfected with thepMACS plasmid encoding either the WT Ptprcb (=CD45.2) or 3 differentmutant versions (with the indicated mutations). An anti-CD45 antibody(recognizing both CD45.2 and CD45.1 isoforms) is used to confirmexpression of the antigen. The WT sequence is CD45.2 positive and CD45.1negative. Only the mutation of the amino acid K in position 277 to Eallows the anti-CD45.1 antibody to bind the antigen and abrogates thebinding of anti-CD45.2 antibody. The other mutations maintain thespecificity for the anti-CD45.2 antibody.

FIG. 7: Gene-editing of the CD45.2 antigen using designer nucleases inorder to abrogate recognition of the antigen by the antigen recognizinganti-CD45.2 antibody. The 1881 cell line naturally expressing CD45.2 wastransfected by co-electroporation with 3 plasmids; one encoding thegreen fluorescent protein (as transfection control), one encoding theguide RNA sequences (gRNA 1 and 2 or for control CD20 gRNA1) (see SEQ IDNO:10 and SEQ ID NO:11, or SEQ ID NO:12 and SEQ ID NO:13) and oneencoding the nuclease Cas9. 4 days after gene-editing, the CD45.1expression of the GFP positive 1881 cells was analyzed by flow cytometryusing the anti-CD45.2 antibody. The 2 dot plots on the right representcontrols as indicated for a sample not incubated with anti-CD45.2antibody.

FIG. 8: represents Table 1 from the manuscript of Holmes (Immunology(2006), vol. 117: 145-155) which details the known (functional) isoformsof human CD45.

DETAILED DESCRIPTION OF THE INVENTION

In targeted therapies including those utilizing antigen recognizingreceptors side-effects resulting from the presence of the target antigenon non-target cells represent a major hurdle for the treatment ofdiseases such as cancer. Such side-effects often include and sometimesare restricted to hematopoietic cells, which leads to the depletion ofone or more hematopoietic subpopulations. The invention is based on thefact that small subsets of hematopoietic cells or the entirehematopoietic system can be replaced through transplantation. If appliedhematopoietic cells are resistant to recognition of saidantigen-recognizing receptor, they can be used to replace said depletedhematopoietic cells and present a way for treatment of the side-effectsof an antigen recognizing receptor. Exemplarily, the concept of thepresent invention is shown by using antigens CD20 and CD45 and selectedantigens thereof. But it is self-explanatory that the concept of thepresent invention is not restricted to these antigens, the selectedvariants thereof, and the cells used herein.

In one aspect the invention provides a system for use in immunotherapyfor reducing the side-effects of an antigen-recognizing receptor againstantigen-expressing non-target cells in an individual, comprising

-   -   a) Said antigen-recognizing receptor wherein said        antigen-recognizing receptor specifically recognizes an antigen        on target cells in said individual;    -   b) Hematopoietic cells resistant to recognition of said antigen        by said antigen-recognizing receptor;        wherein said antigen is expressed on target cells in said        individual and at least on one hematopoietic cell type of said        individual.

In another aspect the invention provides the use of a system for use inimmunotherapy for reducing the side-effects of an antigen-recognizingreceptor against antigen-expressing non-target cells in an individual,the system comprising

-   -   a) Said antigen-recognizing receptor wherein said        antigen-recognizing receptor specifically recognizes an antigen        on target cells in said individual;    -   b) Hematopoietic cells resistant to recognition of said antigen        by said antigen-recognizing receptor;        wherein said antigen is expressed on target cells in said        individual and at least on one hematopoietic cell type of said        individual.

In a further aspect the invention provides a method for reducing theside-effects in an immunotherapy of an antigen-recognizing receptoragainst antigen-expressing non-target cells in an individual, comprising

-   -   a) Application of the antigen-recognizing receptor to said        individual, wherein said antigen recognizing receptor        specifically recognizes an antigen on target cells in said        individual;    -   b) Application of hematopoietic cells resistant to recognition        of said antigen by said antigen-recognizing receptor;        wherein said antigen is expressed on target cells in said        individual and at least on one hematopoietic cell type of said        individual.

In a further aspect the invention provides a method for producing asystem for use in immunotherapy for reducing side-effects of anantigen-recognizing receptor against antigen-expressing non-target cellsin an individual, wherein said system comprises two compositions, themethod comprises

-   -   a) generating an antigen-recognizing receptor wherein said        antigen-recognizing receptor specifically recognizes an antigen        on target cells in said individual, thereby generating the first        composition of said system; and    -   b) generating hematopoietic cells resistant to recognition of        said antigen by said antigen-recognizing receptor, thereby        generating the second composition of said system;        wherein said antigen is expressed on target cells in said        individual and at least on one hematopoietic cell type of said        individual.

The antigen-recognizing receptor may be a soluble receptor or may beexpressed on the cell membrane of an immune effector cell. Cell membranestanding receptors include but are not restricted to natural receptorssuch as T cell receptors (TCR) or genetically engineered receptors suchas transgenic TCRs or chimeric antigen receptors (CAR). Immune effectorcells include but are not restricted to cells with cytotoxic effectorfunction such as natural killer (NK) cells or T cells. Immune effectorcells may comprise one or more cellular subsets. In a preferred variantof the invention the immune effector cell is a T cell engineered toexpress a CAR.

Said hematopoietic cells may be hematopoietic stem cells orhematopoietic progenitor cells.

-   -   Said hematopoietic cells resistant to recognition of said        antigen by said antigen-recognizing receptor may have a        deviation in said antigen, thereby altering the epitope        recognized by said antigen-recognizing receptor resulting in a        non-recognition or alleviated recognition by said        antigen-recognizing receptor.    -   Said immune effector cells expressing said antigen-recognizing        receptor may be said hematopoietic cells resistant to        recognition of said antigen by said antigen-recognizing        receptor, thereby avoiding fratricide.    -   Said immune effector cells expressing said antigen-recognizing        receptor may be autologous or allogeneic transplants.    -   Said hematopoietic cells resistant to recognition of said        antigen by said antigen-recognizing receptor may be autologous        or allogeneic transplants.

Said antigen may be selected from, but not restricted to the groupconsisting of CD11a, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47,CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133,CD135, CD157, CD172b, CD217, CD300a, CD305, CD317 and CD321.

Said antigen-recognizing receptor may comprise an antigen binding domainspecifically recognizing the antigen CD20, and wherein saidhematopoietic cells resistant to recognition of said antigen by saidantigen-recognizing receptor may have a deviation in said antigen,thereby altering the epitope recognized by said antigen-recognizingreceptor.

Said antigen-recognizing receptor may be a CAR, and wherein said antigenbinding domain of said CAR may comprise the amino acid sequences SEQ IDNO:1 (V_(H)) and SEQ ID NO:2 (V_(L)). Said deviation in said CD20antigen of said hematopoietic cells may be the amino acid substitutionalanine to serine at position 170 and/or proline to serine at position172 of said antigen CD20. The amino acid sequence of CD20 is given inSEQ ID NO:3.

The CAR may comprise, from the N-terminus to the C-terminus theextracellular part comprising at least one antigen binding domain, thetransmembrane domain, and the at least one intracellular signalingdomain. But the CAR may also comprise two or more members ofpolypeptides which together work as functional active CAR (e.g.switch-on CAR, conditionally active CAR, regulatable CAR, controllableCAR, multi chain CAR), e.g. a first polypeptide comprising at least oneantigen binding domain, a first member of a dimerization pair, and atransmembrane domain; and a second polypeptide comprising a secondmember of a dimerization pair, and at least one intracellular signalingdomain (for the key signaling of the active CAR), and optionally atransmembrane domain, wherein the CAR is activated upon dimerization bya dimerizer recognizing the members of the dimerization pair. Such CARconstructs with split key signaling and recognition modules aredisclosed e.g. in WO2014/127261A1, WO2015017214A1, WO2015090229A1,WO2015142661A1, and WO2015150771A1. Engineered immune cells,preferentially T cells of the invention express a CAR of the invention,which is able to redirect antigen recognition based on the antigenbinding specificity of the CAR.

The antigen-recognizing receptor of the invention such as a CARrecognizes and binds to the antigen via a certain epitope that composesonly a small fraction of the antigen such as a peptide sequence of aprotein. In turn, deviations in the epitope result in alteration of thebinding affinity of the antigen-recognizing receptor to its antigen andmay lead to the complete loss of specific binding. The inventionexploits the effect that hematopoietic cells expressing a target antigenwith such altered epitope are resistant to recognition and binding ofthe antigen-recognizing receptor of the invention. Such hematopoieticcells may be derived from an individual carrying a naturally occurringpolymorphism that leads to the altered epitope or the altered epitopemay be generated through genetic modification of said hematopoieticcells.

EMBODIMENTS

In one embodiment of the invention the antigen-recognizing receptor inthe present method or system for reducing side-effects of anantigen-recognizing receptor against antigen-expressing non target cellsis a CAR, which is expressed on an immune effector cell, preferentiallyon a T cell. Methods for purification and generating immune effectorcells engineered to express a CAR are well known in the art. Immuneeffector cells, preferentially T cells can be obtained from a variety ofsources including but not restricted to peripheral blood mononuclearcells (PBMCs), leukapheresis or bone marrow samples. For enrichment ofthese cells methods well known in the art can be used such ascentrifugation through a Ficoll™ or PERCOLL™ gradient orpositive/negative selection techniques such as fluorescent sorting (e.g.FACS) or magnetic sorting (e.g. MACS®). In one embodiment T cellsobtained from an individual are magnetically labelled, for example witha magnetic bead coupled to antibodies specific for CD4 and for CD8,respectively, magnetically enriched and collected. In one embodiment theT cells engineered to express a CAR can be activated and expanded prioror after genetic engineering to increase the amount of engineered Tcells generally using methods well known in the art, for example usingpolyclonal stimulation with anti-CD3/anti-CD28 beads oranti-CD3/anti-CD28 nanomatrices (EP2711418A1). Preferentially, saidamount of engineered T cells is increased to a therapeutic effectiveamount.

In one embodiment of the invention an immune effector cell,preferentially a T cell expressing a CAR is generated using methodscommonly known in the art. In a preferred embodiment T cells areengineered to express a CAR using a viral-based system for example alentiviral vector or a γ-retroviral vector in order to achieveexpression of the CAR in said T cells over several weeks, months or upto several years.

In one embodiment of the invention the CAR of the present method orsystem may be specific for the transmembrane protein CD20 that isexpressed on diseased cells in an individual, for example a cancer cellsuch as a CD20 positive melanoma cell or a CD20 positive malignanthematopoietic cell for example a lymphoma cell or a chronic lymphoidleukemia cell or an acute lymphoid leukemia cell.

In one embodiment of the invention the hematopoietic cells resistant torecognition of said antigen by said CAR of the present method or systemmay be hematopoietic stem cells. These cells are altered in respect tosaid antigen for example CD20, so that the altered antigen is notrecognized by said CAR. The deviation of said antigen may be a naturalpolymorphism of said antigen. In one embodiment of the invention saidantigen in said hematopoietic cells, for example hematopoietic stemcells is altered through genetic modification of the genomic DNA of saidhematopoietic cell. The genetic modification may involve the alterationof the genomic DNA sequence encoding said antigen recognized by saidCAR.

Genetic modification of genomic DNA of cells such as hematopoieticcells, for example hematopoietic stem cells can be performed via methodswell known in the art. The genetic modification may be performed byintroduction of designer nucleases such as ZFN, TALEN or CRISPR/Cas intohematopoietic cells. Designer nucleases can be used to introduce strandbreaks at specific locations of the genomic DNA of a cell, which mayinduce error prone repair mechanisms such as non-homologous end joiningin the cell that lead to insertions or deletions at said location of thegenomic DNA. Said location may be part of the genomic sequence encodingfor said antigen and leads to absence of said antigen on said cells. Inone embodiment of the invention a designer nuclease specific for saidantigen, for example CD20 is applied to hematopoietic cells such ashematopoietic stem cells in order to generate a cell that lacks CD20expression. The designer nuclease may be applied together with atemplate DNA in order to exploit the cellular repair mechanism ofhomologous recombination, that leads to inclusion of the genomicsequence of the template DNA at the site of the strand break introducedby the designer nuclease. In one embodiment of the invention a designernuclease specific for the genomic sequence of CD20 is applied tohematopoietic cells such as hematopoietic stem cells, together with atemplate DNA that is largely homologous to the CD20 genomic sequence butcontains one or more sequence alterations.

Both the immune effector cells engineered to express said CAR and thehematopoietic cells with said altered antigen are applied to anindividual suffering from a disease associated with said antigen. In apreferred embodiment of the invention T cells engineered to express aCAR specific for CD20, and hematopoietic stem cells that express analtered version of CD20 not recognized by the CAR, are applied to anindividual suffering from a disease associated with CD20. In oneembodiment of the invention said disease is cancer including but notrestricted to melanoma, lymphoma, chronic lymphoid leukemia or acutelymphoid leukemia. In one embodiment of the invention the individual maybe depleted from hematopoietic cells including hematopoietic stem cellsby a procedure commonly known in the art such as chemotherapy orradiation therapy. In one embodiment of the invention the application ofT cells engineered to express a CAR and hematopoietic cells resistant torecognition of said antigen by said CAR is combined with additionaltreatments. Such treatments may include but are not restricted to theapplication of cytokines or inhibitors of cytokine signaling such asetanercept and tocilizumab.

In a preferred embodiment of the invention immune effector cellsengineered to express said CAR, as well as hematopoietic cells with saidaltered antigen are derived from said individual suffering from adisease associated with said antigen before treatment. Such procedure isknown in the art as an autologous cellular therapy. In anotherembodiment of the invention immune effector cells engineered to expresssaid CAR as well as hematopoietic cells with said altered antigen arederived from one healthy individual and applied to another individualsuffering from a disease associated with said antigen. Such procedure isknown in the art as an allogeneic cellular therapy. The performance ofthe invention in a allogeneic setting may allow the targeting of anantigen with a naturally occurring polymorphism. In one embodiment ofthe invention hematopoietic cells, such as hematopoietic stem cells arederived from an individual with a naturally occurring polymorphism inthe antigen associated with disease. In another embodiment of theinvention hematopoietic cells, such as hematopoietic stem cells as wellas immune effector cells, such as T cells are derived from an individualwith a naturally occurring polymorphism in the antigen associated withdisease. In one embodiment of the invention the natural TCR is depletedfrom such allogeneic T cells using methods commonly known in the art. Inone embodiment of the invention allogeneic immune effector cells may becombined with autologous hematopoietic cells or autologous immuneeffector cells may be combined with allogeneic hematopoietic cells.

The antigen associated with disease may be present on the diseased cellsand otherwise restricted to the immune effector cells engineered, suchas T cells expressing an antigen-recognizing receptor, such as a CAR. Inthat case the side-effects of the antigen recognizing receptor will berestricted to the immune effector cells engineered to express theantigen recognizing receptor. Such side-effect is also known in the artas fratricide. Therefore, in one embodiment of the inventionhematopoietic cells resistant to recognition of said antigen by saidantigen recognizing receptors are said immune effector cells. Suchimmune effector cells may be engineered to express anantigen-recognizing receptor in addition to being genetically modifiedto express an altered version of said antigen. In another embodiment ofthe invention such immune effector cells may be resistant to fratricidedue to the expression of a naturally occurring polymorphic version ofsaid antigen.

Peptides derived from membrane associated, intra-as well asextracellular proteins are presented on the cells surface in complexwith HLA molecules and can be specifically detected by TCRs. SpecificHLA-antigen complexes can also be detected through transgenic TCRs. Inone embodiment the invention provides a method for reducing the sideeffects of an immune effector cell, such as a T cell engineered toexpress a transgenic TCR, by application of hematopoietic cells thatpresent an HLA-antigen complex, that cannot be recognized by saidtransgenic TCR. In one embodiment of the invention said HLA-antigencomplex is formed by said HLA molecule with a peptide comprising analtered sequence, or said HLA-antigen complex is not formed, for exampledue to an altered peptide sequence. The alteration of the peptidesequence may be a natural polymorphism of said peptide or may begenerated through genetic modification. Natural polymorphisms ofpeptides presented on HLA molecules are also known as minor antigens inthe art.

The antigen used in the present method or system has to be expressed ontarget cells in the diseased individual and at least on onehematopoietic cell type. Preferentially, the said antigen is a cellsurface antigen selected from, but not restricted to the groupconsisting of CD11a, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47,CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133,CD135, CD157, CD172b, CD217, CD300a, CD305, CD317 and CD321. Morepreferentially, said antigen is CD20. In one embodiment of the inventionsaid antigen has at least one a splice variant within the extracellulardomain of said antigen suitable for generating different epitopes forantigen binding domains. In another embodiment of the invention saidantigen has at least a natural polymorphic form within the extracellulardomain of said antigen suitable for generating different epitopes forantigen binding domains. In another embodiment of the invention saidantigen can be modified to a non-natural polymorphic form within theextracellular domain of said antigen suitable for generating differentepitopes for antigen binding domains, wherein said non-naturalpolymorphic form does not alter or affect the natural function ofhematopoietic cells in an individual. In another embodiment of theinvention said antigen can be deleted without alteration or affectingthe natural function of hematopoietic cells in said individual.

The antigen-recognizing receptor of the present invention (either insoluble form or as part of an immune cells) may be administered eitheralone, or as a pharmaceutical composition in combination with diluentsand/or with other components such as IL-2 or other cytokines or cellpopulations. Briefly, pharmaceutical compositions of the presentinvention may comprise the antigen-recognizing receptor of the presentinvention as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives.

Preferentially, the compositions of the present invention are formulatedfor intravenous administration. The administration of cell compositionsto the subject may be carried out in any convenient manner known in theart.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease treated. Appropriate dosages maybe determined by clinical trials. The quantity and frequency ofadministration will also be determined and influenced by factors such asthe condition of the patient, and the type and severity of the patient'sdisease.

A pharmaceutical composition comprising immune cells as disclosed hereinmay be administered at a dosage of 10² to 10⁹ cells/kg body weight,preferably 10³ to 10⁶ cells/kg body weight. The cell compositions mayalso be administered several times at these dosages. The compositions ofcells may be injected directly into a tumor, lymph node, or site ofinfection. The cells may be activated and expanded to therapeuticeffective amounts using methods known in the art. The cells of theinvention may be used in combination with e.g. chemotherapy, radiation,immunosuppressive agents, antibodies or antibody therapies.

The hematopoietic cells resistant to recognition of said antigen by saidantigen-recognizing receptor may be administered at the same time or ata time point prior to or after the antigen recognizing receptor of theinvention or the immune effector cells expressing the antigenrecognizing receptor of the invention, including CAR T cells. In apreferred embodiment hematopoietic cells resistant to recognition ofsaid antigen by said antigen-recognizing receptor are administeredbefore treatment with immune effector cells expressing the antigenrecognizing receptor of the invention. Thereby partial or completereconstitution of the blood system with hematopoietic cells resistant torecognition of said antigen by said antigen-recognizing receptor isachieved. For enhanced reconstitution chemotherapy treatment may be usedprior to administration of said hematopoietic cells. In this settingduring treatment with the immune effector cells the treated individualwill always have an at least partially functional blood system, thecomplete depletion of the blood system will be avoided.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The term “diseased cell” as used herein refers to the state of a cell,tissue or organism that diverges from the normal or healthy state andmay result from the influence of a pathogen, a toxic substance,irradiation or cell internal deregulation. “Diseased cell” may alsorefer to a cell that has been infected with a pathogenic virus. Furtherthe term “diseased cell” may refer to a malignant cell or neoplasticcell that may constitute or give rise to cancer in an individual.

The term “cancer” is known medically as a malignant neoplasm. Cancer isa broad group of diseases involving upregulated cell growth. In cancer,cells (cancerous cells) divide and grow uncontrollably, formingmalignant tumors, and invading nearby parts of the body. The cancer mayalso spread to more distant parts of the body through the lymphaticsystem or bloodstream. There are over 200 different known cancers thataffect humans.

The term “malignant” or “malignancy” describes cells, groups of cells ortissues that constitute a neoplasm, are derived from a neoplasm or canbe the origin of new neoplastic cells. The term is used to describeneoplastic cells in contrast to normal or healthy cells of a tissue. Amalignant tumor contrasts with a non-cancerous benign tumor in that amalignancy is not self-limited in its growth, is capable of invadinginto adjacent tissues, and may be capable of spreading to distanttissues. A benign tumor has none of those properties. Malignancy ischaracterized by anaplasia, invasiveness, and metastasis as well asgenome instability. The term “premalignant cells” refer to cells ortissue that is not yet malignant hut is poised to become malignant.

The term “chemotherapy” refers to the treatment of cancer (cancerouscells) with one or more cytotoxic anti-neoplastic drugs(“chemotherapeutic agents” or “chemotherapeutic drugs”) as part of astandardized regimen. Chemotherapy may be given with a curative intentor it may aim to prolong life or to palliate symptoms. It is often usedin conjunction with other cancer treatments, such as radiation therapy,surgery, and/or hyperthermia therapy. Traditional chemotherapeuticagents act by killing cells that divide rapidly, one of the mainproperties of most cancer cells. This means that chemotherapy also harmscells that divide rapidly under normal circumstances, such as cells inthe bone marrow, digestive tract, and hair follicles. This results inthe most common side-effects of chemotherapy, such as myelosuppression(decreased production of blood cells, hence also immunosuppression),mucositis (inflammation of the lining of the digestive tract), andalopecia (hair loss).

The term “immune cell” or “immune effector cell” refers to a cell thatmay be part of the immune system and executes a particular effectorfunction such as alpha-beta T cells, NK cells, NKT cells, B cells,innate lymphoid cells (ILC), cytokine induced killer (CIK) cells,lymphokine activated killer (LAK) cells, gamma-delta T cells,mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes ormacrophages. Preferred immune cells are cells with cytotoxic effectorfunction such as alpha-beta T cells, NK cells, NKT cells, ILC, CIKcells, LAK cells or gamma-delta T cells. “Effector function” means aspecialized function of a cell, e.g. in a T cell an effector functionmay be cytolytic activity or helper activity including the secretion ofcytokines.

The term “side-effects” refers to any complication, unwanted orpathological outcome of an immunotherapy with an antigen recognizingreceptor that occurs in addition to the desired treatment outcome. Theterm “side-effect” preferentially refers to on-target off-tumortoxicity, that might occur during immunotherapy in case of presence ofthe target antigen on a cell that is an antigen-expressing non-targetcell but not a diseased cell as described herein. A side-effect of animmunotherapy may be the developing of graft versus host disease.

The term “reducing side-effects” refers to the decrease of severity ofany complication, unwanted or pathological outcome of an immunotherapywith an antigen recognizing receptor such as toxicity towards anantigen-expressing non-target cell. “Reducing side-effects” also refersto measures that decrease or avoid pain, harm or the risk of death forthe patient during the immunotherapy with an antigen recognizingreceptor.

The term “combination immunotherapy” refers to the concerted applicationof two therapy approaches e.g. therapy approaches known in the art forthe treatment of disease such as cancer. The term “combinationimmunotherapy” may also refer to the concerted application of animmunotherapy such as the treatment with an antigen recognizing receptorand another therapy such as the transplantation of hematopoietic cellse.g. hematopoietic cells resistant to recognition by the antigenrecognizing receptor.

Expression of an antigen on a cell means that the antigen is sufficientpresent on the cell surface of said cell, so that it can be detected,bound and/or recognized by an antigen-recognizing receptor.

The term “hematopoietic cells”, refers to a population of cells of thehematopoietic lineage capable of hematopoiesis which include but is notlimited to hematopoietic stem cells and/or hematopoietic progenitorcells (i.e., capable to proliferate and at least partially reconstitutedifferent blood cell types, including erythroid cells, lymphocytes, andmyelocytes). The term “hematopoietic cells” as used herein also includesthe cells that are differentiated from the hematopoietic stem cellsand/or hematopoietic progenitor cells to form blood cells (i.e. bloodcell types, including erythroid cells, lymphocytes, and myelocytes).

A hematopoietic cell resistant to recognition of an antigen by anantigen-recognizing receptor means that said cell cannot as easily bedetected, bound and/or recognized by an antigen-recognizing receptorspecific for said antigen or that the detection, binding and/orrecognizing is impaired, so that no or reduced side-effects can beobserved during a immunotherapy using the cells of the presentinvention.

An antigen is said to be “resistant” to recognition by a receptor or anantibody if it is expressed in a form, in a manner, or at a density thatinhibits or reduces binding of the receptor. Either it binds at least10-fold (and preferably 10-fold to 1000-fold, more preferably 1000 foldto 100,000 fold and most preferably 100,000 to 10,000,000 fold) lessreceptor under the same conditions as the wild-type antigen does whenpresented under normal conditions, or it binds the receptor with atleast 10-fold (and preferably 10-fold to 1000-fold, more preferably 1000fold to 100,000 fold and most preferably 100,000 to 10,000,000 fold)lower affinity. In certain embodiments of the invention, the antigen isrecombinantly modified to inhibit or reduce receptor binding. Typically,an antigen modified in this way is over 90% identical to the wild typeisoform at the amino acid level, but has been recombinantly introducedwith at least one, two, three, five, or more than five altered aminoacids, additions, or amino acid deletions that change primary,secondary, or tertiary protein structure so as to inhibit or reducereceptor recognition. In other cases the antigen can be modified (e.g.shortened, elongated or truncated) to eliminate the binding motif.However the original functionality of the target antigen is preferablynot affected by the modification.

By way of illustration, the hematopoietic cell resistant to recognitionof said antigen by an antigen-recognizing receptor specific for saidantigen which is used in the method or system of the present inventioncan be generated for example by using an antigen

-   -   1) which has at least one splice variant within the        extracellular domain of said antigen suitable for generating        different epitopes for antigen binding domains, or    -   2) which has at least a natural polymorphic form within the        extracellular domain of said antigen suitable for generating        different epitopes for antigen binding domains; or    -   3) which can be modified to a non-natural polymorphic form        within the extracellular domain of said antigen suitable for        generating different epitopes for antigen binding domains,        wherein said non-natural polymorphic form does not alter or        affect the natural function of said antigen on the cells; or    -   4) which can be deleted (e.g. knocked out) without alteration or        affecting the natural function of the hematopoietic cell.

The result is a deviation in said antigen so that the hematopoietic cellcannot be recognized by said antigen-recognizing receptor anymore or canbe recognized in a alleviated form only.

The term “fratricide” refers to the observation that the antigenassociated with disease may be, in addition to diseased cells, presenton immune effector cells engineered, such as T cells expressing anantigen-recognizing receptor, such as a CAR. In that case theside-effects of the antigen recognizing receptor will affect the immuneeffector cells engineered to express the antigen recognizing receptor.Such side-effect is also known in the art as fratricide.

In general, the term “receptor” refers to a biomolecule that may besoluble or attached to the cell surface membrane and specifically bindsa defined structure that may be attached to a cell surface membrane orsoluble. Receptors include but are not restricted to antibodies andantibody like structures, adhesion molecules, transgenic or naturallyoccurring TCRs or CARS. In specific, the term “antigen-recognizingreceptor” as used herein may be a membrane bound or soluble receptorsuch as a natural TCR, a transgenic TCR, a CAR, a scFv or multimersthereof, a Fab-fragment or multimers thereof, an antibody or multimersthereof, a bi-specific T cell enhancer (BiTE), a diabody, or any othermolecule that can execute specific binding with high affinity.

The term “antigen” refers to a molecular entity that may be soluble orcell membrane bound in particular but not restricted to molecularentities that can be recognized by means of the adaptive immune systemincluding but not restricted to antibodies or TCRs, or engineeredmolecules including but not restricted to transgenic TCRs, CARS, scFvsor multimers thereof, Fab-fragments or multimers thereof, antibodies ormultimers thereof, single chain antibodies or multimers thereof, or anyother molecule that can execute binding to a structure with highaffinity.

The term “target” or “target antigen” refers to any cell surfaceprotein, glycoprotein, glycolipid or any other structure present on thesurface of the target cell. The term also refers to any other structurepresent on target cells in particular but not restricted to structuresthat can be recognized by means of the adaptive immune system includingbut not restricted to antibodies or TCRs, or engineered moleculesincluding but not restricted to transgenic TCRs, CARS, scFvs ormultimers thereof, Fab-fragments or multimers thereof, antibodies ormultimers thereof, single chain antibodies or multimers thereof, or anyother molecule that can execute binding to a structure with highaffinity.

The term “target cells” as used herein refers to cells which arerecognized by the antigen-recognizing receptor which is or will beapplied to the individual.

The term “antigen-expressing non-target cell” as used herein refers tothe healthy cells (non-diseases cells) of an individual treated with themethod of the present invention, which express the same antigen as thetarget cells. The recognition of said antigen on the antigen-expressingnon-target cells is not desired and may lead to complication(side-effects) on treatment in state of the art immunotherapy.

The terms “specifically binds” or “specific for” or “specificallyrecognize” with respect to an antigen-recognizing receptor refer to anantigen-binding domain of said antigen-recognizing receptor whichrecognizes and binds to a specific antigen, but does not substantiallyrecognize or bind other molecules in a sample. An antigen-binding domainthat binds specifically to an antigen from one species may bind also tothat antigen from another species. This cross-species reactivity is notcontrary to the definition of that antigen-binding domain as specific.An antigen-binding domain of an antigen-recognizing receptor thatspecifically binds to an antigen may bind also to different allelicforms of the antigen (allelic variants, splice variants, isoforms etc.).This cross reactivity is not contrary to the definition of thatantigen-binding domain as specific as long as these different allelicforms of the antigen do not take part in the intended derivation of theantigen needed for the generation of the resistance of the hematopoieticcells to the antigen as used in the present invention.

The term “system for use in immunotherapy” as used herein refers to theconstellation that two kinds of compositions are needed to perform thecombined immunotherapy as disclosed herein. Therefore, the system (orset or kit or the combination of compositions) comprises

-   -   a) an antigen-recognizing receptor wherein said        antigen-recognizing receptor specifically recognizes an antigen        on target cells in said individual;    -   b) hematopoietic cells resistant to recognition of said antigen        by said antigen-recognizing receptor.

“Chimeric antigen receptor” or “CAR” refer to engineered receptors,which graft an antigen specificity onto cells, for example T cells. TheCARs of the invention comprise an antigen binding domain also known asantigen targeting region, an extracellular spacer domain or hingeregion, a transmembrane domain and at least one intracellular signalingdomain or a least one co-stimulatory domain and at least oneintracellular signaling domain.

In general, a CAR may comprise an extracellular domain (extracellularpart) comprising the antigen binding domain, a transmembrane domain andan intracellular signaling domain. The extracellular domain may belinked to the transmembrane domain by a linker. The extracellular domainmay also comprise a signal peptide.

A “signal peptide” refers to a peptide sequence that directs thetransport and localization of the protein within a cell, e.g. to acertain cell organelle (such as the endoplasmic reticulum) and/or thecell surface.

An “antigen binding domain” refers to the region of the CAR thatspecifically binds to an antigen (and thereby is able to target a cellcontaining an antigen). The CARS of the invention may comprise one ormore antigen binding domains. Generally, the targeting regions on theCAR are extracellular. The antigen binding domain may comprise anantibody or a fragment thereof. The antigen binding domain may comprise,for example, full length heavy chain, Fab fragments, single chain Fv(scFv) fragments, divalent single chain antibodies or diabodies. Anymolecule that binds specifically to a given antigen such as affibodiesor ligand binding domains from naturally occurring receptors may be usedas an antigen binding domain. Often the antigen binding domain is ascFv. Normally, in a scFv the variable portions of an immunoglobulinheavy chain and light chain are fused by a flexible linker to form ascFv. Such a linker may be for example the “(G₄/S₁)₃-linker”.

In some instances, it is beneficial for the antigen binding domain to bederived from the same species in which the CAR will be used in. Forexample, when it is planned to use it therapeutically in humans, it maybe beneficial for the antigen binding domain of the CAR to comprise ahuman or humanized antibody or fragment thereof. Human or humanizedantibodies or fragments thereof can be made by a variety of methods wellknown in the art.

“Spacer” or “hinge” as used herein refers to the hydrophilic regionwhich is between the antigen binding domain and the transmembranedomain. The CARs of the invention may comprise an extracellular spacerdomain but is it also possible to pass such a spacer. The spacer mayinclude Fc fragments of antibodies or fragments thereof, hinge regionsof antibodies or fragments thereof, CH2 or CH3 regions of antibodies,accessory proteins, artificial spacer sequences or combinations thereof.A prominent example of a spacer is the CD8alpha hinge.

The transmembrane domain of the CAR can be derived from any desirednatural or synthetic source for such domain. When the source is naturalthe domain may be derived from any membrane-bound or transmembraneprotein. The transmembrane domain may be derived for example fromCD8alpha or CD28. When the key signaling and antigen recognition modulesare on two (or even more) polypeptides then the CAR may have two (ormore) transmembrane domains. Splitting key signaling and antigenrecognition modules enables for a small molecule-dependent, titratableand reversible control over CAR cell expression (Wu et al, 2015, Science350: 293-303) due to small molecule-dependent heterodimerizing domainsin each polypeptide of the CAR.

The cytoplasmic domain or the intracellular signaling domain of the CARis responsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR is expressed. “Effectorfunction” means a specialized function of a cell, e.g. in a T cell aneffector function may be cytolytic activity or helper activity includingthe secretion of cytokines. The intracellular signaling domain refers tothe part of a protein which transduces the effector function signal anddirects the cell expressing the CAR to perform a specialized function.

The intracellular signaling domain may include any complete or truncatedpart of the intracellular signaling domain of a given protein sufficientto transduce the effector function signal. Prominent examples ofintracellular signaling domains for use in the CARs include thecytoplasmic sequences of the T cell receptor (TCR) and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement.

Generally, T cell activation can be mediated by two distinct classes ofcytoplasmic signaling sequences, firstly those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences) and secondly those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences, costimulatorysignaling domain). Therefore, an intracellular signaling domain of a CARmay comprise a primary cytoplasmic signaling domain and/or a secondarycytoplasmic signaling domain.

Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain ITAMs (immunoreceptor tyrosine-based activation motifssignaling motifs). Examples of ITAM containing primary cytoplasmicsignaling sequences often used in CARS are that are those derived fromTCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma , CD3 delta , CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequencederived from CD3 zeta.

The cytoplasmic domain of the CAR can be designed to comprise theCD3-zeta signaling domain by itself or combined with any other desiredcytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise aCD3 zeta chain portion and a costimulatory signaling region. Thecostimulatory signaling region refers to a part of the CAR comprisingthe intracellular domain of a costimulatory molecule. A costimulatorymolecule is a cell surface molecule other than an antigen receptor ortheir ligands that is required for an efficient response of lymphocytesto an antigen. Examples for a costimulatory molecule are CD27, CD28,4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.The cytoplasmic signaling sequences within the cytoplasmic signalingpart of the CAR may be linked to each other in a random or specifiedorder. A short oligo- or polypeptide linker, which is preferably between2 and 10 amino acids in length, may form the linkage. A prominent linkeris the glycine-serine doublet.

As an example, the cytoplasmic domain may comprise the signaling domainof CD3-zeta and the signaling domain of CD28. In another example thecytoplasmic domain may comprise the signaling domain of CD3-zeta and thesignaling domain of CD27. In an further example, the cytoplasmic domainmay comprise the signaling domain of CD3-zeta, the signaling domain ofCD28, and the signaling domain of CD27.

As aforementioned either the extracellular part or the transmembranedomain or the cytoplasmic domain of a CAR may also comprise aheterodimerizing domain for the aim of splitting key signaling andantigen recognition modules of the CAR.

The CAR of the invention may be designed to comprise any portion or partof the above-mentioned domains as described herein in any combinationresulting to a functional CAR.

To qualify as a “chimeric antigen receptor” in the embodiments of theinvention claimed below, a CAR minimally has at least anantigen-specific variable region (typically a single chain variableregion comprised of antibody heavy and light chain variable regions)linked to a T cell signaling domain: typically an intracellular domainof a T-cell receptor, exemplified by (but not limited to) the zetadomain of CD3. Upon binding of the antigen-specific region to thecorresponding antigen, the T cell signaling domain mediates a T cellfunction in the host cell (such as cytotoxicity). The CAR may optionallybut does not necessarily comprise additional domains, such as a linker,a transmembrane domain, and other intracellular signaling elements asdescribed above.

The term “genetic modification” or genetically modified” refers to thealteration of the nucleic acid content including but not restricted tothe genomic DNA of a cell. This includes but is not restricted to thealteration of a cells genomic DNA sequence by introduction exchange ordeletion of single nucleotides or fragments of nucleic acid sequence.The term also refers to any introduction of nucleic acid into a cellindependent of whether that leads to a direct or indirect alteration ofthe cells genomic DNA sequence or not.

The terms “engineered cell” and “genetically modified cell” as usedherein can be used interchangeably. The terms mean containing and/orexpressing a foreign gene or nucleic acid sequence, which in turnmodifies the genotype or phenotype of the cell or its progeny.Especially, the terms refer to the fact that cells can be manipulated byrecombinant methods well known in the art to express stably ortransiently peptides or proteins, which are not expressed in these cellsin the natural state. Genetic modification of cells may include but isnot restricted to transfection, electroporation, nucleofection,transduction using retroviral vectors, lentiviral vectors,non-integrating retro- or lentiviral vectors, transposons, designernucleases including zinc finger nucleases, TALENs or CRISPR/Cas.

The term “therapeutic effective amount” means an amount, which providesa therapeutic benefit.

Immunotherapy is a medical term defined as the “treatment of disease byinducing, enhancing, or suppressing an immune response”. Immunotherapiesdesigned to elicit or amplify an immune response are classified asactivation immunotherapies, while immunotherapies that reduce orsuppress are classified as suppression immunotherapies. Cancerimmunotherapy as an activating immunotherapy attempts to stimulate theimmune system to reject and destroy tumors. Adoptive cell transfer usescell-based cytotoxic responses to attack cancer cells. Immune cells suchas T cells that have a natural or genetically engineered reactivity to apatient's cancer are generated in vitro and then transferred back intothe cancer patient.

The term “treatment” as used herein means to reduce the frequency orseverity of at least one sign or symptom of a disease.

As used herein, the term “individual” refer to an animal.Preferentially, the individual is a mammal such as mouse, rat, cow, pig,goat, chicken dog, monkey or human. More preferentially, the individualis a human. The individual may be an individual suffering from a diseasesuch as cancer (a patient), but the subject may be also a healthysubject.

The amino acid sequences of anti-human CD20 V_(H), anti-human CD20V_(L), are given in SEQ ID NO:1 and SEQ ID NO:2, respectively (in theone-letter code of amino acids). The amino acid sequences (proteins,polypeptides) as given in the SEQ ID NO:1 and SEQ ID NO:2 refer to allconstellations of the respective amino acid sequence which retains theintended function of the respective amino acid sequence as definedherein. In other words, the divergences to the SEQ ID No:1 and SEQ IDNO:2, respectively, should not affect their potential as bindingspecifically to the antigen CD20 and/or being a functional CAR.Therefore, the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 canbe the full length amino acid sequence of the SEQ ID NO:1 and SEQ IDNO:2, respectively. It can also be a variant thereof which have someamino acids deleted, added or replaced while still retaining theintended function as described herein. Therefore, included in thisdefinition are variants of the amino acid sequences in SEQ ID NO: 1 andSEQ ID NO:2, respectively, such as amino acid sequences essentiallysimilar to SEQ ID NO: 1 and SEQ ID NO:2, respectively, having a sequenceidentity of at least 70%, or at least 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% at the amino acid sequence level. In the context of the presentinvention, “sequence identity” may be determined using pairwisealignments using alignments programs for amino acid sequences well knownto the art.

EXAMPLES Example 1: Targeting the CD20 Antigen With a CD20-RecognizingCAR Expressed in T cells Where 2 Amino Acids of the Wild Type CD20Antigen Have Been Mutated to Abrogate CD20 Recognition by the AntigenRecognizing Receptor

In a first instance we have used HEK 293 T cells to overexpress severalvariants of the human CD20 antigen (the sequences were synthesized byDNA 2.0 and cloned into the plasmid pMACS LNGFR-IRES of MiltenyiBiotec). FIG. 2 shows how mutations of amino acids A into S at position170 and P into S at positions 172 of the wild type human CD20 antigen(SEQ ID NO:3) abrogate binding of the anti-CD20 antibody clone 2H7.

A CAR encoding the antigen-recognizing receptor of the 2H7 (see SEQ IDNO 1 and 2) with an IgG1 extracellular spacer, a CD8 transmembrane andan intracellular signaling domain of 4-1BB-CD3 zeta was generated andcloned into a lentiviral vector (provided by Lentigen Technology, Inc.,USA). Human T cells were activated with MACS GMP TransAct kit (MiltenyiBiotec according to the manufacturers recommendations, transduced withthe CD20 CAR encoding lentiviral vector at a multiplicity of infectionof 2 and expanded in TexMACS medium with 20 ng/ml human IL-2 (MiltenyiBiotec) for 14 days. The CAR transduced T cells where then co-culturedfor 24 hours at a ratio of 1 to 1 with the target cells indicated inFIG. 2. FIG. 3 shows the measurements of GM-CSF and IL-2 expressed bythe CART cells in the supernatant of the culture (using the MACSPlex™cytokine measurement system of Miltenyi Biotec). The CAR T cellsspecifically produce cytokines in the presence of HEK-293 T cells thatexpress the wild type CD20 antigen but not when the HEK-293 T cellsexpress the mutant CD20 antigen.

Thus, using minor gene-editing, it is possible to maintain expression ofthe CD20 antigen while abrogating recognition by the T cells expressingthe antigen recognizing receptor.

Several methods can be used to edit genes. In order to illustrate thepossibility to switch human CD20 wild type expression to expression ofthe mutant form, we have electroporated the Raji cell line (whichnaturally expresses wild type CD20) with the Cas9 nuclease, theindicated guide RNA (see FIG. 4 and SEQ ID NO:4 to SEQ ID NO:9) and atransfection control encoding GFP using the plasmids. FIG. 4 shows thatthe best “loss” of CD20 APC staining was obtained when using the gRNA2(15% of the GFP transfected cells).

FIG. 4 represents the proof of principle that an epitope of an antigenexpressed by cells can be altered. Thus a target cell can become anon-target for a given antigen-recognizing receptor expressed by a giveneffector cell.

In this example, the antigen CD20 is not expressed by the T cellstherefore no gene-editing of the antigen in the T cells would benecessary in order to avoid fratricide. In this application,hematopoietic stem cells (of the same T cell donor) would however beedited (similarly to FIG. 4) in order for the progeny of the HSCs to notbe recognized by the CD20 CAR T cells. Thus the patient with wild typeCD20 positive target cells such as tumor cells but also healthy B cellsexpressing the wild type CD20 would be depleted of all original CD20positive cells (tumor and healthy B cells) but healthy B cells withmutated CD20 antigen would be able to repopulate the patient thereforereducing sustained B cell aplasia.

Example 2: Use of an Antigen Recognizing Receptor to Target an AntigenPresent on the Majority of Haematopoietic Cells Including T Cells

In this example we have taken advantage of the polymorphism of CD45 inmouse models. As indicated in FIG. 5, the amino acid differences guidingthe polymorphism of CD45.1 and CD45.2 isoforms in C57BL/6 mice is known.One can generate antibodies specifically recognizing the 2 isoformsseparately. Such antibodies exist that recognize either the CD45.2 orthe CD45.1 isoform of the mouse CD45 surface molecule. However, in thismodel it is unknown, which epitopes the existing antibodies recognize.We have therefore used plasmids encoding the sequence of three differentmutants of the CD45.2 isoform or the wild type CD45.2 to forceexpression of said antigens in the HEK-293T cell line. As indicated inFIG. 6, mutation of the amino acid K in position 277 to E allows theanti-CD45.1 antibody to bind the antigen and abrogates the binding ofanti-CD45.2 antibody. The 2 other constructs maintain specificity forthe CD45.2 antibody.

Therefore it is possible to use the anti-CD45.2 antibody to generate ananti-CD45.2 recognizing CAR that would be “blind” to the K in position277 to E mutation, or vice versa to use the anti-CD45.1 antibody togenerate an anti-CD45.1 recognizing CAR that would be “blind” to the Kin position 277.

Similarly as in example 1 and as shown in FIG. 7, the CD45.2 expressedat the surface of the 1881 cell line could be, in part, gene-edited withthe gRNA SEQ ID NO:12 and SEQ ID NO:13 and the cas9 nuclease to not berecognized by the CD45.2 antibody.

FIG. 8 represents known polymorphisms of the human CD45 antigen. Severalforms are associated with disease while others are not.

Example 3: Editing of Antigens Present in Malignancies Associated toEarly Precursors of HSCs and/or HSCs

In this example the antigens associated to the disease are also presenton healthy HSCs but not on T cells. Polymorphisms of antigens such asCD34 or CD133 are defined, and antigen-recognizing receptors are definedthat can specifically target the wild type antigens but not specificmutations of the wild type antigen. As in example 3, healthy HSCs arepurified (e.g. using a cell sorter and markers capable ofdifferentiating healthy HSCs from the diseased or the malignant ones),gene edited and infused into the patient. T cells from the same patientare then modified to express the antigen-recognizing receptor withoutthe need to be edited for the antigen.

Sequence Listing

heavy chain variable domain (V_(H)) of anti-human CD20 SEQ ID NO: 1MAQVKLQESG AELVKPGASV KMSCKASGYT FTSYNMHWVKQTPGQGLEWI GAIYPGNGDT SYNQKFKGKA TLTADKSSSTAYMQLSSLTS EDSADYYCAR SNYYGSSYWF FDVWGQGTTV TVSSlight chain variable domain (V_(L)) of anti-human CD20 SEQ ID NO: 2DIELTQSPTI LSASPGEKVT MTCRASSSVN YMDWYQKKPGSSPKPWIYAT SLASGVPARF SGSGSGTSYS TISRVEAEDA ATYYCQQWSF NPPTFGGGTK LEIKwild-type human CD20 SEQ ID NO: 3MTTPRNSVNG TFPAEPMKGP IAMQSGPKPL FRRMSSLVGPTQSFFMRESK TLGAVQIMNG LFHIALGGLL MIPAGIYAPICVTVWYPLWG GIMYIISGSL LAATEKNSRK CLVKGKMIMNSLSLFAAISG MILSIMDILN IKISHFLKME SLNFIRAHTPYINIYNCEPA NPSEKNSPST QYCYSIQSLF LGILSVMLIFAFFQELVIAG IVENEWKRTC SRPKSNIVLL SAEEKKEQTIEIKEEVVGLT ETSSQPKNEE DIEIIPIQEE EEEETETNFP EPPQDQESSP IENDSSPTarget site 1 Rev G19nGG (CD20 gRNA1) Oligo Fw SEQ ID NO: 4ACCGATGG GGAGTTTTTC TCAGAGTarget site 1 Rev G19nGG (CD20 gRNA1) Oligo Rev SEQ ID NO: 5AAAACTCTGA GAAAAACTCC CCATCGTarget site 2 Rev G19nGG (CD20 gRNA2) Oligo Fw SEQ ID NO: 6ACACCGTAAC AGTATTGGGT AGATGGTarget site 2 Rev G19nGG (CD20 gRNA2) Oligo Rev SEQ ID NO: 7AAAACCATCT ACCCAATACT GTTACGTarget site 5 Rev G17nGG (CD20 gRNA3) Oligo Fw SEQ ID NO: 8ACACCGTATG CTGTAACAGT ATTGTarget site 5 Rev G17nGG (CD20 gRNA3) Oligo Rev SEQ ID NO: 9AAAACAATAC TGTTACAGCA TACGTarget site 1 Rev G18nGG (CD45.2 gRNA1) Oligo Fw SEQ ID NO: 10ACACCGTTGC ATTTTCTGAA ATCAGTarget site 1 Rev G18nGG (CD45.2 gRNA1) Oligo Rev SEQ ID NO: 11AAAACTGATT TCAGAAAATG CAACGTarget site 2 Fw G19nGG (CD45.2 gRNA2) Oligo Fw SEQ ID NO: 12ACACCGGCTA ATACTTCAAT TTGTTGTarget site 2 Fw G19nGG (CD45.2 gRNA2) Oligo Rev SEQ ID NO: 13AAAACAACAA ATTGAAGTAT TAGCCG

While the invention has been described with reference to the specificembodiments, changes can be made and equivalents can be substituted toadapt to a particular context or intended use, thereby achievingbenefits of the invention without departing from the scope of what isclaimed.

1. A product combination that is configured for removing target cellsfrom a subject, wherein the combination comprises the following: (a) afirst cell population containing immune effector cells that express ontheir surface a recombinant antigen-recognizing receptor thatspecifically recognizes and binds a target antigen that is present onthe target cells in the subject and on at least some blood cells in thesubject that are not target cells, wherein binding of the immuneeffector cells to the target antigen on the target cells results inremoval of a plurality of the target cells; and (b) a second cellpopulation containing hematopoietic cells that have a gene that encodesa form of target antigen that is different from the form of the targetantigen that is present on the target cells, whereby blood cells thatare progeny of the hematopoietic cells that express the target antigenwill express it in the different form, wherein the different form of thetarget antigen is resistant to recognition by the antigen-recognizingreceptor that is expressed by the first cell population.
 2. The productcombination of claim 1, wherein the different form of the target antigenexpressed by the second cell population is a splice variant or anaturally occurring polymorphic form of the target antigen.
 3. Theproduct combination of claim 1, wherein the different form of the targetantigen expressed by the second cell population contains a recombinantlyintroduced polymorphism that generates a different epitope within theextracellular domain of the target antigen without altering function ofthe second cell population.
 4. The product combination of claim 1,wherein the different form of the target antigen expressed by the secondcell population contains one or more amino acid deletions that result inthe antigen-recognizing receptor having reduced binding affinity for thetarget antigen.
 5. The product combination of claim 1, wherein thedifferent form of the target antigen expressed by the second cellpopulation contains a plurality of amino acid substitutions that resultin the antigen-recognizing antigen receptor having reduced bindingaffinity for the target antigen.
 6. The product combination of claim 1,wherein hematopoietic cells in the second cell population have beengenetically altered so that an endogenous gene that encodes the targetantigen encodes it in the different form.
 7. The product combination ofclaim 1, wherein the first cell population is a population of Tlymphocytes that have been genetically modified so that they express ontheir surface the recombinant antigen-recognizing receptor that bindsthe target antigen on the target cells.
 8. The product combination ofclaim 6, wherein the antigen-recognizing receptor expressed on thesurface of the first cell population is a chimeric antigen receptor(CAR) comprising an antibody variable region specific for the targetantigen in tandem with a T cell stimulatory domain.
 9. The productcombination of claim 1, wherein the target cells are cancer cells. 10.The product combination of claim 1, wherein the target cells are a typeof hematopoietic cell that expresses the target antigen.
 11. The productcombination of claim 1, wherein the target antigen is CD20.
 12. Theproduct combination of claim 11, wherein the antigen-recognizingreceptor comprises SEQ. ID NO:1 (V_(H)) and SEQ. ID NO:2 (V_(L)). 13.The product combination of claim 1, wherein the antigen-recognizingreceptor expressed by the first cell population has an affinity for thetarget antigen expressed in the altered form by the second cellpopulation that is 10-fold to 1000-fold less than its affinity for thetarget antigen as it is expressed on the target cells.
 14. The productcombination of claim 1, wherein the first cell population is autologousto the subject.
 15. The product combination of claim 1, wherein thefirst cell population and the second cell population are both autologousto the subject.
 16. The product combination of claim 1, provided in theform of a single composition that contains both the first cellpopulation and the second cell population.
 17. The product combinationof claim 1, wherein the first cell population and the second cellpopulation are separate, but packaged together as a kit.
 18. A methodfor preparing the product combination according to claim 1, comprising:(a) obtaining T lymphocytes from the subject; (b) forming the first cellpopulation by a process that comprises genetically modifying the Tlymphocytes obtained in step (a) so that they express on their surface arecombinant antigen-recognizing receptor specific for an antigen that ispresent on target cells in the subject and on at least some blood cellsin the subject that are not target cells; (c) obtaining hematopoieticcells from the subject; and (d) forming the second cell population by aprocess that comprises genetically modifying the hematopoietic cellsobtained in step (c) such that blood cells expressing the target antigenthat are progeny of the hematopoietic cells will express it in analtered form that is resistant to recognition by the antigen-recognizingreceptor expressed by the first cell population.